Abstract

The eukaryotic initiation factor 4A (eIF4A) is a DEAD box helicase that unwinds RNA structure in the 5' untranslated region (UTR) of mRNAs. Here, we investigated the role of eIF4A in porcine sapovirus VPg-dependent translation. Using inhibitors and dominant-negative mutants, we found that eIF4A is required for viral translation and infectivity, suggesting that despite the presence of a very short 5' UTR, eIF4A is required to unwind RNA structure in the sapovirus genome to facilitate virus translation.

The PSaV genome contains stable stem-loops at the 5′ end. (A) Secondary structure in the 5′ end of the PSaV RNA genome predicted by Mfold (). (B) SHAPE analysis of in vitro-transcribed PSaV genome (GIII, pCV4A[9]) performed as described in the text. Modified bases are indicated with an arrow. Numbers refer to nucleotide positions. The locations of the start codon and the full length are highlighted. Primers binding to nucleotides (nt) 123 to 142 (left) and 246 to 265 (right) were used. Strong bands present on both gels are marked by asterisks for reference. The positions of modified bases are marked with a black dot in panel A. (C) Predicted secondary RNA structures of Sapovirus genogroups I, II, IV, and V (Dresden, AY694184; Mc10, AY237420; Ehime 1107, DQ058829; and NK, AY646856) in schematic representation were analyzed using Mfold (). Minimum free energies for the first stem, containing the AUG (box), in each structure are indicated as ΔG.

PSaV translation is inhibited by hippuristanol and eIF4A dominant-negative mutants. In vitro translation was performed using either VPg-linked PSaV RNA (40 ng/μl) or dicistronic RNA (20 ng/μl) containing a cap-dependent CAT and PTV IRES-dependent luciferase (Fluc). Translation reaction mixtures were preincubated with increasing concentrations of hippuristanol (A), recombinant eIF4AI wt (B), or dominant-negative mutants (C to E). RNAs were then added to initiate protein synthesis. The products for VPg-, cap-, and IRES-dependent translations were resolved by SDS-PAGE and visualized by autoradiography. The intensity of each band was quantitated with reference to the value obtained in the absence of hippuristanol, wt eIF4AI, or dominant-negative mutants as appropriate.

eIF4A binds PSaV mRNA during virus replication. (A) Viral RNA was coimmunoprecipitated from PSaV-infected cells with anti-VPg, anti-eIF4A, or anti-IgG antibodies. Immunoprecipitated RNA was quantified by quantitative PCR (qPCR) with primers specific for PSaV protease and is presented as a percentage relative to the total amount of input lysates. (B) Interaction between PSaV (1 μM) or GAPDH RNA (4 μM) and 10 μM eIF4AI or eIF4G-C fragment in the presence of AMP-PNP (2 mM). The His tag pulldown assay was subsequently performed with recombinant His-tagged eIF4AI or eIF4G-C. Relative RNA binding was calculated upon addition of AMP-PNP. (C) Relative RNA binding in the presence and absence of hippuristanol was assayed as in panel B. All experiments were performed three times, and the results are expressed as means ± standard errors of the means (SEM): **, P < 0.005; ***, P < 0.0001.

eIF4A is required for efficient PSaV replication in cell culture. (A) PSaV infectivity was determined by 50% tissue culture infective dose (TCID50) after treatment with MeSO4 or increasing concentrations of hippuristanol. (B) Transfection with either control or eIF4AI small interfering RNAs (siRNAs) was performed in LLC-PK cells. Decreased eIF4AI protein expression was verified by Western blotting prior to PSaV infection. (C) PSaV mRNA was quantitated by qPCR after control or eIF4AI siRNA transfection. (D) The levels of infectious virus were determined by titer by TCID50. Samples were analyzed in triplicate in three independent experiments. Error bars represent the means ± SEM from triplicate samples: *, P < 0.05; ***, P < 0.0001. The dashed line is used to indicate the limit of detection by TCID50.